Semi-continuous blood separation using magnetic beads

ABSTRACT

A container having a flexible wall receives a continuous flow of a biological fluid that includes a target antigen and emits a continuous flow of the biological fluid at least partially depleted from the target antigen. The container further comprises a compartment with magnetic beads coupled to an affinity marker that binds the target antigen, and the target antigen is separated from the biological fluid using a magnetic force and an automatic mechanical force, wherein at least one of the magnetic force and automatic mechanical force is transmitted through the flexible wall.

[0001] This application is a C-I-P of U.S. patent application Ser. No.09/949,314, filed Sep. 7, 2001, which is a C-I-P of U.S. patentapplication Ser. No. 09/514,686, now U.S. Pat. No. 6,291,249, which is aC-I-P of U.S. patent application Ser. No. 09/261,068 filed Mar. 2, 1999abandoned Jul. 31, 2000, all of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

[0002] The field of the invention is blood separation.

BACKGROUND OF THE INVENTION

[0003] Separation, and especially on line separation of target antigensfrom biological fluids for diagnostic and/or curative purposes is oftendesirable, especially where the biological fluid is a whole blood orother bodily fluid. Among frequently isolated and/or separated targetantigens are diseased cells (e.g., T4-helper cells infected with HIVvirus), non-diseased cells (e.g., adult stem cells), various bacteria,and viruses.

[0004] There are numerous methods of on line separation or isolation oftarget antigens known in the art, and such methods may be grouped intoone of two categories. For example, in a discontinuous separation, abiological fluid is isolated from a source (e.g., blood is drawn from ahuman) and subsequently processed for the desired target antigen. Mostcommonly, such methods include centrifugation for relatively largevolumes of biological fluids, or magnetic separation for relativelysmall volumes. In either case, the processed fluid may then bereintroduced into the human. While discontinuous separation is generallyrelatively effective, various disadvantages remain. Among other things,processing of the isolated biological fluid typically requires transferof the fluid among a multitude of containers, which significantlyincreases the likelihood of contamination of the processed sample.Furthermore, depending on the particular the volume of the isolatedfluid, multiple samples need to be drawn for sufficient amount of targetantigen.

[0005] On the other hand, in a continuous separation, a biological fluidis directly routed from a source (e.g., via i.v. line) into a separationunit and continuously processed for the desired target antigen beforethe depleted fluid is rerouted into the source. Most commonly, suchmethods include gradient centrifugation, or on line magnetic separation.While discontinuous separation is generally advantageous with respect tosimplified sample handling, all or almost all of the on line separationsrequire relatively expensive and intricate equipment. Consequently, manycontinuous separation methods and configurations present variouschallenges. For example, continuous centrifugation equipment (e.g., asthe equipment used in thrombocytophoresis) typically requires control byhighly trained professionals. For magnetic separation, cells or othertarget antigens are separated by virtue of their magnetic moment,thereby typically limiting separation efficiency at desirable flowrates.

[0006] Although various configurations and methods for isolation and/orseparation of one or more components from blood are known to the art,all or almost all of them suffer from one or more disadvantage.Therefore, there is a need to provide apparatus and methods for improvedisolation and/or separation of components from blood.

SUMMARY OF THE INVENTION

[0007] The present invention is directed to apparatus and methods for acontainer having at least one flexible wall, a fluid receiving port, afluid discharge port, and a plurality of compartments fluidly coupled toat least one of the fluid receiving port and the fluid discharge port.The fluid receiving port in contemplated containers receives acontinuous flow of a biological fluid that includes a target antigen,and the fluid discharge port emits a continuous flow of the biologicalfluid that is at least partially depleted from the target antigen,wherein at least one of the compartments further comprises a pluralityof magnetic beads that are coupled to an affinity marker that binds thetarget antigen, and wherein the target antigen is separated from thebiological fluid using a magnetic force and an automatic mechanicalforce, wherein at least one of the magnetic force and automaticmechanical force is transmitted through the flexible wall.

[0008] In one aspect of the inventive subject matter, one or more of thecompartments include a buffer, a wash fluid, an isotonic fluid, and/oran elution fluid, and at least one of the compartments may furtherinclude a port that allows draining of the least one of the compartment.

[0009] In a further aspect of the inventive subject matter, the affinitymarker is selected from the group consisting of an antibody, an antibodyfragment, and a lectin, and particularly preferred biological fluidscomprise whole blood. It is further preferred that the target antigen isselected from the group consisting of a stem cell, a diseased cell, abacterium, and a virus.

[0010] Various objects, features, aspects and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention, along with theaccompanying drawing.

DETAILED DESCRIPTION

[0011] It is generally contemplated that a target antigen is separatedand/or isolated from a biological fluid in a container with a flexiblewall in a semi-continuous fashion using magnetic beads via an automatedmechanical force and a magnetic force.

[0012] More particularly, it is contemplated that a container has atleast one flexible wall, a fluid receiving port, a fluid discharge port,and a plurality of compartment fluidly coupled to at least one of thefluid receiving port and the fluid discharge port, wherein the fluidreceiving port receives a continuous flow of a biological fluid thatincludes a target antigen, and wherein the fluid discharge port emits acontinuous flow of the biological fluid that is at least partiallydepleted from the target antigen, wherein at least one of thecompartments further comprises a plurality of magnetic beads that arecoupled to an affinity marker that binds the target antigen, and whereinthe target antigen is separated from the biological fluid using amagnetic force and an automatic mechanical force, wherein at least oneof the magnetic force and automatic mechanical force is transmittedthrough the flexible wall. As used herein, the term “continuous flow”refers to the flow of the fluid in and out of the container duringoperation, and includes any flow rate above 0.1 ml/min. Typicalcontinuous flow rates are generally in the range between 1-100 ml/min,but may also be less than 1 ml/min (e.g., 0.1 ml/min to 1 ml/min), oreven higher than 100 ml/min, especially where the container isrelatively large. In less preferred aspects, however, it is contemplatedthat the flow may also be discontinuous (i.e., the flow rate may beintermittently below 0.1 ml/min).

[0013] The general concept of magnetic separation using an automatedmechanical force and a magnetic force is disclosed in commonly ownedU.S. Pat. No. 6,291,249 and U.S. patent application Ser. No. 09/261,068filed Mar. 2, 1999, both of which are incorporated herein by reference.FIG. 1 depicts an exemplary container 100 having a flexible top sheet102 laminated to a back sheet (not shown). The term “flexible” as usedherein refers to a quality of material characterized in that thematerial can be deformed to a significant degree without destruction.For example, a soft plastic foil is considered flexible, while a hardplastic or glass plate is not considered flexible under the scope ofthis definition. A fluid receiving port 110 that receives the biologicalfluid is fluidly coupled to compartments 130 and 132, which are in turnfluidly coupled to separation chamber 160. Separation chamber 160includes a plurality of magnetic beads 170, and at least some of thebeads are coated with an affinity marker (not shown). Separation chamber160 is further fluidly coupled to the following compartments:Compartment 140 (e.g., containing wash fluid), compartment 150 (e.g.,containing elution fluid), compartment 142 (e.g., for receiving wastefluid), compartment 152 (e.g., for receiving eluted target antigen), andcompartments 134 and 136. Compartments 134 and 136 are further fluidlycoupled to fluid discharge port 120. Additional ports AP1 and AP2 arefluidly coupled to compartment 142 and 152, respectively, and allowdraining of the fluid and/or target antigen from the compartments.

[0014] With respect to the container 100 it should be appreciated thatthe container may be fabricated from various materials. For example, itis generally preferred that the container is fabricated from a flexibleback sheet and a flexible top sheet, and that all compartments and fluidconduits that fluidly couple one compartment to another compartment areformed by the top and bottom sheet. However, in alternative aspects, theback sheet may be inflexible, while only the top sheet is flexible.Moreover, it should be appreciated that while it is preferred that theentire top sheet is flexible, in alternative containers only portions ofthe top sheet are flexible. In still further contemplated aspects, atleast a portion of the top sheet and/or bottom sheet may be transparent(e.g., to perform optical measurements, including scatter, absorptionand/or transmission). Thus, at least one of the compartments and theconduits will include a flexible portion through which flow of the fluidin the compartment and/or conduit can be controlled, initiated, orstopped by pressing an actuator or other mechanical device thereon.However, it is generally preferred that the entire container isflexible, and may therefore be stored in a roll or otherwise non-flatconfiguration.

[0015] It should further be appreciated that the volume of the variouscompartments in contemplated containers may vary considerably, and aparticular volume will generally depend on the particular separationand/or isolation procedure. For example, where the target antigen is aHepatitis C virus (frequently present at relatively high loads), thevolume of the compartments may be between about 1-3 ml (or less) andabout 4-20 ml, and more. On the other hand, where the target antigen isa particular set of leukocytes, suitable volumes of the compartments maybe between about 5-10 ml (or less) and about 20-100 ml, and more. Stillfurther, it should be recognized that where volumes of particular fluidsare exceeding the volume of the appropriate compartment, additionalports (see e.g., AP1 and AP2) may be included through which excessvolume my be drained or added.

[0016] Furthermore, it is contemplated that while at least some of thecompartments may comprise a fluid (which may be employed to wash,dilute, and/or elute a target antigen from the magnetic bead), othercompartments may be empty prior to operation of the container. Forexample, it is preferred that the compartment 130, 142, 152, and 134 areempty to receive the biological fluid when the process is initiated.Preferred containers may further include adhesive labels, permanentlabels, bar codes, etc., to encode test and patient data, and may haveadditional channels or other means for proper alignment with aninstrument that receives the container and delivers the automaticmechanical force and the magnetic force.

[0017] It is further contemplated that the conduits that fluidly couplethe compartments are configured such that an actuator or othermechanical device may compress the conduit to partially or completelystop flow of the fluid through the conduit. In preferred containers, atleast one, more typically several, and most typically all of the fluidconduits are at least temporarily sealable (i.e., flow of the fluidthrough the conduit can be stopped). Alternatively, or additionally,contemplated conduits may include jet valves, Venturi valves, or othermeans that restrict the flow of fluid in a unidirectional manner, orallow flow of fluid only above a predetermined pressure. There arenumerous such means known in the art, and all of those are contemplatedsuitable for use herein.

[0018] With respect to the magnetic beads, it is preferred that suchbeads comprise a paramagnetic composition embedded in synthetic polymersor cellulose. Although paramagnetic particles are preferred, the coatedparticles can also or alternatively include ferromagnetic or chromiummaterial or mixtures thereof. In still further variations, suitableparticles can be coated with many other materials including natural orsynthetic polymers, agarose etc. The preferred particle size is in therange of 0.1-100 micrometers, but alternative sizes between 10-100micrometers or larger than 100 micrometers are also contemplated. Viewedfrom another aspect, it is contemplated to employ particles having amean volume between about 5×10⁻²⁴m³ and about 5×10⁻⁶m³. The term“coated” is used herein to mean any complete or partial covering of anyexposed surface. It should further be appreciated that by employingmagnetic beads in a compartment that can be fluidly isolated from othercompartments, separation of the target antigen bound to the magneticbead will occur in a quasi-stationary manner. Viewed from anotherperspective, it should be appreciated that the separation usingcontemplated containers is not based on a separation using a magneticmoment of a magnetic particle in a flow of a biological fluid.

[0019] In further preferred aspects of the inventive subject matter,contemplated beads are coated with polyclonal or monoclonal antibodiesor antibody fragments that have a binding specificity towards the targetantigen. Therefore, suitable antibodies or antibody fragmentsparticularly include commercially available preparations. However, itshould be recognized that contemplated affinity markers may also includenon-protein affinity molecules and may therefore include nucleic acids,biotin, lectins, etc. Thus, contemplated target antigens includeproteins, nucleic acids, avidin-labeled samples, viruses, bacteria, andhealthy (stem cells) and diseased cells (infected cells).

[0020] Consequently, it is contemplated that depending on the particularnature of the target antigen at least one of the compartments (e.g.,152) may operate as a compartment to isolate a target antigen, or toseparate a target antigen for further reaction. For example, it iscontemplated that in such a compartment various biochemical reactionsmay be performed. Especially contemplated reactions include chromogenicreactions and PCR (with predispensed reagents in the compartment).Therefore, it should also be recognized that at least one compartment ofthe container may be heated, cooled, agitated, or illuminated.

[0021] In an exemplary operation, it is contemplated that a continuousflow of a biological fluid is provided (e.g., via an i.v. line poweredby a peristaltic pump) to the fluid receiving port 110 and compartment130, both of which will act as a temporary reservoir for the incomingbiological fluid, while a predetermined amount of a predispensed bufferor biological fluid previously directed to compartment 132 is directed(e.g., via actuators or other hydraulic pressure) to the separationcompartment that includes a plurality of magnetic beads to which anaffinity marker is coupled. Once compartment 132 is (at least partially)emptied, compartment 132 may act (optionally in conjunction with FRP110) as a temporary reservoir for the incoming biological fluid, and thebiological fluid in compartment 130 is directed towards the separationcompartment.

[0022] It is further contemplated that the target antigen in the fluidin compartment 160 will bind to the affinity markers 170 on the magneticbeads, and it is still further contemplated that the magnetic beads (andwith this the target antigens) will be retained compartment 160 by amagnet (in a position proximal to compartment 160, not shown) while theremainder of the biological fluid is passed on to at least one ofcompartment 134 and 136. In an optional wash step, the target antigenmay now be washed with a wash fluid from compartment 140 and the spentwash fluid may then be directed to a waste compartment 142. The targetantigen may then be removed from the magnetic beads 170 using an elutionfluid from compartment 150, and the eluted target antigen is collectedin compartment 152. It should be particularly appreciated that all ofthe target antigen separation and/or isolation steps may be performedwhile compartment 110 and at least one of the compartments 130 and 132continuously receive the biological fluid.

[0023] Once the biological fluid (now depleted from the target antigen)is directed to 134 and/or 136, a new separation cycle may be initiated.Furthermore, it should be appreciated that by employing at least 2compartments (here: 134 and 136) in fluid communication with the fluiddischarge port 120, a continuous discharge of depleted biological fluidis achieved. For example, while the depleted fluid from 136 and 120 isdischarged (e.g., directed back to the source of the biological fluid),compartment 134 can be filled at the same time, and will thereforeprovide continuous flow of depleted biological fluid once 136 is (atleast partially) emptied. Alternatively, additional compartments may beincluded to receive a fluid within the container as temporaryreservoirs.

[0024] With respect to the automatic mechanical force, it is generallypreferred that at least one, and more preferably a plurality ofactuators that compress respective compartments to provide actuation ofthe fluid. It is further preferred that at least one, and more typicallysome or all of the conduits that fluidly couple the compartments will beconfigured such that an actuator pressing on such a conduit will preventflow of the fluid from one compartment to another compartment. Stillfurther it is contemplated that one or more compartments may include aport or opening that allows draining or refill of the compartment.

[0025] Thus, specific embodiments and applications of semi-continuousblood separation using magnetic beads have been disclosed. It should beapparent, however, to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended contemplated claims. However, it should be appreciated thatclaims in a non-provisional application based on this provisionalapplication may be entirely different or different in part. Moreover, ininterpreting the specification, all terms should be interpreted in thebroadest possible manner consistent with the context. In particular, theterms “comprises”, and “comprising”, should be interpreted as referringto elements, components, or steps in a non-exclusive manner, indicatingthat the referenced elements, components, or steps may be present, orutilized, or combined with other elements, components, or steps that arenot expressly referenced.

What is claimed is:
 1. An apparatus comprising: a container having atleast one flexible wall, a fluid receiving port, a fluid discharge port,and a plurality of compartments fluidly coupled to at least one of thefluid receiving port and the fluid discharge port; wherein the fluidreceiving port receives a continuous flow of a biological fluid thatincludes a target antigen, and wherein the fluid discharge port emits acontinuous flow of the biological fluid that is at least partiallydepleted from the target antigen; wherein at least one of thecompartments further comprises a plurality of magnetic beads that arecoupled to an affinity marker that binds the target antigen; and whereinthe target antigen is separated from the biological fluid using amagnetic force and an automatic mechanical force, wherein at least oneof the magnetic force and automatic mechanical force is transmittedthrough the flexible wall.
 2. The apparatus of claim 1 wherein at leastone of the compartments includes a fluid selected from the groupconsisting of a buffer, a wash fluid, an isotonic fluid, and an elutionfluid.
 3. The apparatus of claim 1 wherein the affinity marker isselected from the group consisting of an antibody, an antibody fragment,and a lectin.
 4. The apparatus of claim 1 wherein at least one of thecompartments further includes a port that allows draining of the leastone of the compartments.
 5. The apparatus of claim 1 wherein thebiological fluid comprises whole blood.
 6. The apparatus of claim 1wherein the target antigen is selected from the group consisting of astem cell, a diseased cell, a bacterium, and a virus.